A fundamental aspect of the cellular response to hormonal and other stimuli is the activation of arachidonic acid metabolism. Many current studies focus on the mechanism of release of free arachidonic acid. This is generally considered to be essential to the activation of the cyclo-oxygenase and lipoxygenase pathways of metabolism. However we believe there is one specific case in which this assumption is not warranted. In our opinion there is a distinct possibility that an arachidonate ester is the substrate of the 5-lipoxygenase of leukocytes. There are aspects of leukotriene synthesis via the 5-lipoxygenase which differ markedly from prostaglandin biosynthesis and the other lipoxygenase pathways. In essence, leukocytes will not readily convert exogenous arachidonic acid to leukotrienes. Even after addition of an appropriate stimulus (ionophore A23187) there is evidence that the endogenous substrate is used preferentially. A straight forward, and experimentally untested, explanation for these findings is that free arachidonic acid is not the 5-lipoxygenase substrate. We propose to test the hypothesis that there can be direct oxygenation of esterfied forms of arachidonate in leukocytes. The hypothesis will be tested by four approaches. We will activate leukocytes with A23187 and use specifically designed chromatographic methods to isolate oxygenated esters (with rigorous controls for autoxidation and re-esterification of products). Secondly we will synthesize radiolabeled esters and examine their metabolism in leukocytes. We will use both intact cells (with delivery of radiolabeled substrates in liposomes) and lyzed cells (with emulsified substrate). Thirdly we will use H2(18)0 media to permit mass spectrometric analysis of hydrolysis/esterification cycles in the turnover of arachidonate. With this technique we will establish if esterification is an obligatory step in the conversion of exogenous arachidonic acid to 5-lipoxygenase products in leukocytes. Finally, in order to substantiate a precedent for our hypothesis of lipoxygenase-catalyzed oxygenation of esters, we will analyze the structures of products from a recently described reaction of soybean lipoxygenase-1 with polyunsatured fatty acids esterified in phosphatidylcholine. Leukotrienes are potent mediators of inflammatory reactions. Unravelling of the initial step in their biosynthesis is essential to our understanding of these processes. The possibility that the 5-lipoxygenase utilizes an arachidonate ester substrate has received no prior experimental examination. Such a finding would add a new dimension to our concepts of activation of arachidonate metabolism.